This project aims at developing and validating diagnostic tools for early viral haemorrhagic fever outbreak detection and control. A frontline line-assay carrying antigens of all African haemorrhagic fever viruses is to be developed and validated for widespread use in local hospitals. Line assays are 3rd generation immunoblots using purified recombinant proteins sprayed onto membranes irrespective of their molecular weight. This means that highly concentrated protein can be arranged onto membranes in the manner of a barcode. There are no problems with contaminating extra bands, or with spacing of target molecules. This assay is easy to use and the results are easy to interpret. It is also cheap to produce and has a long shelf life. This tool will help to improve outbreak detection in Africa. The project includes a long-term field trial in local hospitals. A mobile integrated fluorescent real-time-PCR (F-RT-PCR) for outbreak investigation teams is also to be developed. It will consist of a panel of F-RT-PCRs covering all haemorrhagic fever viruses, a robust simple nucleic acid extraction protocol and ready to use lyophilised PCR mixes. This tool will help outbreak investigation teams to improve their ability to manage and control haemorrhagic fever outbreaks.

Background and Project Objectives:

Control of VHF outbreaks critically depends on early detection, and an early alert, to allow, to define, and deliver an appropriate response. To improve the early detection of cases, adequate tools have to be developed to allow early detection in the basic (field) conditions of local hospitals. Once the outbreak is identified, case management also needs on-site tools such as viral genome detection to contain the spread of the outbreak by carefully identifying and monitoring viraemic patients able to transmit the virus. The general objective of the present project is to make adequate tools available to identify VHF outbreaks on-site at an early stage, and to support and complement the control of an outbreak. To reach this general objective, we are developing a) line assays (LA) for antibody detection as an easy to use frontline detection assay for health care workers in local hospitals, and b) F-RT-PCR assays to be used by specialised mobile outbreak investigation teams that can be used at the scene of the outbreak. Both assays will cover Ebola-Virus (EBOV), Marburg Virus (MRGV), Crimean-Congo-Virus (CCHFV) Lassa virus (LASV), Rift Valley Fever Virus (RVFV), Yellow Fever virus (YFV) and Dengue virus 1-4 (DENV). The F-RT-PCR will additionally cover the most important viral differentials Influenza A virus (FLUAV) and Influenza B virus (FLUBV).

Results

Line Assay: The prototype line assay carrying 7 antigens was successfully developed by the consortium partners. It is currently in the first step of the evaluation consisting of a proficiency trial among the project participants. For the second validation step international reference laboratories have already agreed to test confirmed positive samples from their repositories.

The third validation step consists of an extended 6-month field trial in 24 local hospitals of the African partner countries. This trial will start in April 2010.

F-RT-PCR: Assays for EBOV, MBGV, CCHFV, RVFV, DENV, FLUAV, FLUBV were developed and tested on the mobile Smart Cycler. The mobile cycler has already been in use in the recent DENV outbreak on Carpe Verde and Senegal.